Response element compositions and assays employing same

ABSTRACT

DNA segments have been discovered, and characterized by sequence, which are response elements operative to confer responsiveness to ligands for several members of the steroid/thyroid superfamily of receptors, for the transcriptional activation and/or repression of promoters in cells. By using transcriptional control regions comprising response elements of the present invention in combination with a functional promoter, it is now possible to provide recombinant DNA vectors containing a gene, the transcription (and, thereby, also expression) of which is under the control of a promoter, the transcriptional activity of which is responsive to ligands for members of the steroid/thyroid superfamily of receptors.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 438,757, filed Nov. 16, 1989, now pending, the entire contents ofwhich are hereby incorporated by reference herein.

[0002] This invention was made with Government support awarded by theUnited States National Institutes of Health. The United StatesGovernment has certain rights in the invention.

FIELD OF INVENTION

[0003] The present invention relates to the superfamily of nuclearreceptors known as the steroid/thyroid hormone receptors and theircognate response elements. More particularly, the present inventionrelates to the discovery of novel response elements which may be used tocontrol the transcriptional activity of promoters.

BACKGROUND OF THE INVENTION

[0004] A central question in eukaryotic molecular biology is howspecific DNA-binding proteins bind regulatory sequences to influencecell function and fate. The steroid/thyroid hormone receptors form asuperfamily of ligand-dependent transcription factors that are believedto play a part in such cell function and fate. For example, it is knownthat these receptors transduce extracellular hormonal signals to targetgenes that contain specific enhancer sequences (referred to ashormone-response elements, or HRES). Each receptor contains aligand-binding domain and a DNA-binding domain. The receptor undergoes aconformational change when it binds ligand. This conformational changepermits the receptor-ligand complex to bind its cognate response elementand thereby regulate transcriptional activity of an associated promoter.Transcriptional activation of promoter drives transcription of anoperatively associated structural gene.

[0005] Sequence comparison and mutational analyses of hormone receptors,such as the glucocorticoid receptor (GR), have identified functionaldomains responsible for transcriptional activation and repression,nuclear localization, DNA binding, and hormone binding. The DNA bindingdomain, which is required in order to activate transcription, consistsof 66-68 amino acids of which about 20 sites, including nine cysteines(C₁ to C₉), are invariant among different receptors. The modularstructure of members of this receptor superfamily allows the exchange ofone domain for another to create functional, chimeric receptors.

[0006] The hormone response elements identified thus far are generallystructurally related, but they are in fact functionally distinct. Theresponse elements for GR [i.e., the glucocorticoid response element(GRE)], estrogen receptor [i.e., the estrogen response element (ERE)],and thyroid hormone receptor [i.e., the thyroid hormone responseelements (TREs)] have been characterized in detail; they each consist ofa palindromic pair of ‘half sites’ [Evans, Science 240, 889 (1988);Green and Chambon, Trends in Genetics 4, 309 (1988)]. With optimizedpseudo- or consensus response elements, only two nucleotides per halfsite are different in GRE and ERE [Klock, et al., Nature 329, 734(1987)]. On the other hand, identical half sites can be seen in ERE andTRE, but their spacing is different [Glass, et al., Cell 54, 313(1988)]. Moreover, TRE has been shown to mediate transcriptionalactivation by transfected retinoic acid receptors (RARs) in CV-1 cellswhereas non-transfected cells show no response [Umesono et al., Nature336, 262 (1988)]. In other words, both TR and RAR receptors can activateTREs.

[0007] Thus far, however, the response elements for only a few membersof the steroid/thyroid superfamily of receptors have been identified.The response elements for many other members of the superfamily, and therelationship between them, if any, remain to be described.

SUMMARY OF THE INVENTION

[0008] We have discovered, and characterized by sequence, DNA segmentswhich are response elements operative to confer responsiveness toligands for several members of the steroid/thyroid superfamily ofreceptors, for the transcriptional activation and/or repression ofpromoters in cells. We have also discovered that the transcriptionalactivity modulating effect of the invention response elements occurs inall mammalian cells in the presence of ligands for several members ofthe steroid/thyroid superfamily of receptors, indicating that thevarious hormone receptors recognized by the invention response elementsare present endogenously in all of these cells.

[0009] Contrary to what has previously been reported in the art for theGRE, ERE and TRE, the novel response elements disclosed herein have atandem repeat sequence, as opposed to a palindromic sequence which haspreviously been reported for GRE, ERE and TRE. In addition, theinvention response elements are much less susceptible to transcriptionalactivation by non-cognate receptors than are the previously describedresponse elements (GRE, ERE, TRE).

[0010] By using transcriptional control regions comprising responseelements of the present invention and a functional promoter, it is nowpossible to provide recombinant DNA vectors containing a gene, thetranscription (and, thereby, also expression) of which is under thecontrol of a promoter, the transcriptional activity of which isresponsive to (and modulated by) ligands for several members of thesteroid/thyroid superfamily of receptors.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 represents the sequence of the mouse βRAR promoter regionand the first exon. The TATA and -AGGTCA- motifs are underlined; thefirst exon splice site is indicated with an arrow.

[0012]FIG. 2(a) presents the in vivo analysis of RARβ RA responseelement sequences, following a series of deletions from the 5′-end ofthe sequence including the β retinoic acid response element.

[0013]FIG. 2(b) presents sequences of oligonucleotides including the βretinoic acid response element used in the experiments described herein.

[0014]FIG. 3 presents the sequence for several hormone response elementsand summarizes the responsiveness of such sequences to triiodothyronine(T₃) and retinoic acid (RA).

[0015]FIG. 4 illustrates the interconversion of the MHC-TRE into anRARE.

[0016]FIG. 5 illustrates the selective transactivation of syntheticdirect repeat hormone response elements by vitamin D₃ (VD₃),triiodothyronine (T₃), and retinoic acid (RA).

DETAILED DESCRIPTION OF THE INVENTION

[0017] In accordance with the present invention, there is provided asubstantially pure DNA having the sequence:

5′-RGBNNM-[(N)_(x)-RGBNNM]_(y)-3′,

[0018] wherein

[0019] each R is independently selected from A or G;

[0020] each B is independently selected from G, C, or T;

[0021] each N is independently selected from A, T, C, or G; and

[0022] each M is independently selected from A or C; with the provisothat at least 4 nucleotides of each-RGBNNM-group of nucleotides areidentical with the nucleotides at comparable positions of the sequence-AGGTCA-,

[0023] x is zero or a whole number falling in the range of 2 up to 15,and

[0024] y is at least 1.

[0025] Alternatively, the invention response elements can be describedas substantially pure DNA having the sequence:

5′-AGGTCA-[(N)_(x)-AGGTCA)_(y)-3′,

[0026] wherein N, x and y are as defined above, and one or two of thenucleotides of each -AGGTCA- group of nucleotides can be replaced with adifferent nucleotide, consistently with the definitions provided above,i.e., the first nucleotide can be replaced with a G; the thirdnucleotide of the group can be replaced with C or T; the fourthnucleotide of the group can be replaced with A, G, or C; the fifthnucleotide of the group can be replaced with A, G, or T; and the sixthnucleotide of the group can be replaced with C.

[0027] In accordance with another embodiment of the present invention,there are provided DNA constructs comprising the above-describedresponse elements operatively linked to a promoter which is not normallysubject to transcriptional activation and/or repression by ligands formembers of the steroid/thyroid superfamily of receptors; wherein the DNAand the promoter are operatively linked so as to confer transcriptionalactivation and/or repression activity on said promoter in the presenceof a suitable ligand and its associated receptor. The above-describedconstructs can then be operatively linked to a gene for transcription.The resulting gene-containing DNA construct can then be incorporatedinto a vector for expression. The resulting vector can then betransformed into suitable host cells.

[0028] Cells containing the above-described vectors can then be used forthe controlled expression of a gene of interest, in response to thepresence or absence of a suitable ligand and its associated receptor.

[0029] In accordance with yet another embodiment of the presentinvention, there is provided a method for testing the activity of a testcompound as an agonist of a ligand for a member of the steroid/thyroidsuperfamily of receptors (to which the invention response elementsrespond), said method comprising:

[0030] (a) culturing a cell (as described above) in the presence of amember of the steroid/thyroid superfamily of receptors, and in thefurther presence, or in the absence, of the test compound; andthereafter

[0031] (b) comparing the amount of the protein of interest expressedduring the culturing in the presence, or in the absence, of the testcompound.

[0032] In accordance with still another embodiment of the presentinvention, there is provided a method for testing the activity of a testcompound as an antagonist of a ligand for a member of thesteroid/thyroid superfamily of receptors (to which the inventionresponse elements respond), said method comprising:

[0033] (a) culturing a cell (as described above) in the presence of amember of the steroid/thyroid superfamily of receptors and a ligand forsaid receptor to which the response elements of the present inventionrespond, and further:

[0034] (i) in the presence of the test compound, or

[0035] (ii) in the absence of the test compound; and thereafter

[0036] (b) comparing the amount of the protein of interest expressedduring the (i) and (ii) culturing steps.

[0037] In accordance with a further embodiment of the present invention,there is provided a method to distinguish whether or not responsivenessto a ligand for a first member of the steroid/thyroid superfamily ofreceptors occurs via a pathway unique to at least one member of thesteroid/thyroid superfamily, relative to other member(s) of thesteroid/thyroid superfamily, said method comprising:

[0038] contacting a vector containing an invention response element (asdescribed above) with a ligand for said first member of thesteroid/thyroid superfamily of receptors, and varying ratios ofexpression vectors for a first and second receptor, and thereafter

[0039] determining the effect of increasing ratios of the first receptorexpression vector to the second receptor expression vector ontranscription activation and/or repression of said response element bysaid ligand for said first member of the steroid/thyroid superfamily ofreceptors.

[0040] In accordance with another aspect of the present invention, thereis provided a method to screen compounds to identify those compoundswhich act as ligands for members of the steroid/thyroid superfamily ofreceptors, said method comprising:

[0041] contacting said compound with cells (as described above), whereinsaid cells are further transfected with an expression vector for saidmember of the steroid/thyroid superfamily of receptors, wherein saidreceptor, in the presence of its cognate ligand, is capable of bindingto response elements of the present invention, and thereafter

[0042] assaying for the modulation of expression of the reporterprotein.

[0043] In the present specification and claims, reference is made tophrases and terms of art which are expressly defined for use herein asfollows:

[0044] “RARβ” or “βRAR” both refer to retinoic acid receptor beta;

[0045] “VDRE” means vitamin D3 response element;

[0046] “TRE” means thyroid hormone response element;

[0047] “T₃” means triiodothyronine;

[0048] “CAT” means chloramphenicol acetyl transferase;

[0049] “LUC” means firefly luciferase;

[0050] “β-Gal” means β-galactosidase;

[0051] “COS” means monkey kidney cells which express T antigen (Tag)[see, for example, Gluzman in Cell, 23: 175 (1981)];

[0052] “CV-1” means mouse kidney cells from the cell line referred to as“CV-1”. CV-1 is the parental line of COS. Unlike COS cells, which havebeen transformed to express SV40 T antigen (Tag), CV-1 cells do notexpress T antigen;

[0053] “transcriptional control region” or “transcriptional controlelement” refer to a DNA segment comprising a response elementoperatively linked to a promoter to confer ligand responsiveness totranscriptional activity of the promoter;

[0054] “operatively linked” means that the linkage (i.e., DNA segment)between the DNA segments so linked is such that the described effect ofone of the linked segments on the other is capable of occurring.Effecting operable linkages for the various purposes stated herein iswell within the skill of those of ordinary skill in the art,particularly with the teaching of the instant specification;

[0055] “promoter being naturally unresponsive to ligand” means thatligand does not enhance transcription from the promoter to an observableextent in a cell (e.g., a mammalian cell) unless a response element ofthe invention is spliced or inserted (upstream of the promoter) relativeto the direction of transcription therefrom, by recombinant DNA orgenetic engineering methods, into a DNA segment comprising the promoter,and linked to the promoter in a manner which makes transcriptionalactivity from the promoter operatively responsive to ligand;

[0056] “substantial sequence homology” refers to DNA or RNA sequenceswhich have de minimus sequence variations from, and retain the samefunctions as, the actual sequences disclosed and claimed herein;

[0057] “members of the steroid/thyroid superfamily of receptors” refersto hormone binding proteins that operate as ligand-dependenttranscription factors, including identified members of thesteroid/thyroid superfamily of receptors for which specific ligands havenot yet been identified (referred to hereinafter as “orphan receptors”).Each such protein has the intrinsic ability to bind to a specific DNAsequence in a target gene. Following binding, the transcriptionalactivity of the gene is modulated by the presence or absence of thecognate hormone (ligand). The DNA-binding domains of all of thesenuclear receptors are related, consisting of 66-68 amino acid residues,and possessing about 20 invariant amino acid residues, including ninecysteines. A member of the superfamily can be identified as a proteinwhich contains these diagnostic amino acid residues, which are part ofthe DNA-binding domain of such known steroid receptors as the humanglucocorticoid receptor (amino acids 421-486), the estrogen receptor(amino acids 185-250), the mineralocorticoid receptor (amino acids603-668), the human retinoic acid receptor (amino acids 88-153). Thehighly conserved amino acids of the DNA-binding domain of members of thesuperfamily are as follows:

Cys-X-X-Cys-X-X-Asp*-X-Ala*-X-Gly*-X-Tyr*-X-X-X-X-Cys-X-X-Cys-Lys*-X-Phe-Phe-X-Arg*-X-X-X-X-X-X-X-X-X-(X-X-)Cys-X-X-X-X-X-(X-X-X-)Cys-X-X-X-Lys-X-X-Arg-X-X-Cys-X-X-Cys-Arg*-X-X-Lys*-Cys-X-X-X-Gly*-Met;

[0058] wherein X designates non-conserved amino acids within theDNA-binding domain; the amino acid residues denoted with an astericksare residues that are almost universally conserved, but for whichvariations have been found in some identified hormone receptors; and theresidues enclosed in parenthesis are optional residues (thus, theDNA-binding domain is a minimum of 66 amino acids in length, but cancontain several additional residues). Examplary members of thesteroid/thyroid superfamily of receptors include steroid receptors suchas glucocorticoid receptor, mineralocorticoid receptor, progesteronereceptor, androgen receptor, vitamin D₃ receptor, and the like; plusretinoid receptors, such as RARα, RARβ, RARγ, and the like; thyroidreceptors, such as TRα, TRβ, and the like; as well as other geneproducts which, by their structure and properties, are considered to bemembers of the superfamily, as defined hereinabove. Examples of orphanreceptors include HNF4 [see, for example, Sladek et al., in Genes &Development 4: 2353-2365 (1990)], the COUP family of receptors [see, forexample, Miyajima et al., in Nucleic Acids Research 16: 11057-11074(1988), Wang et al., in Nature 340: 163-166 (1989)], COUP-like receptorsand COUP homologs, such as those described by Mlodzik et al., in Cell60: 211-224 (1990) and Ladias et al., in Science 251: 561-565 (1991),the ultraspiracle receptor-[see, for example, Oro et al., in Nature 347:298-301 (1990)], and the like;

[0059] “suitable ligands” for hormone receptors of the steroid/thyroidsuperfamily refers to the specific ligand(s) which, in combination withits cognate receptor, is effective to transcriptionally activate theresponse element to which the cognate receptor binds (i.e., RA/RAR/RARE,vitamin D₃/vitamin D₃ receptor/VDRE, T₃/TR/TRE, estrogen/ER/ERE, and thelike).

[0060] The nucleotides which occur in the various nucleotide sequencesappearing herein have their usual single-letter designations (A, G, T, Cor U) used routinely in the art.

[0061] In the present specification and claims, references to Greekletters may either be written out as alpha, beta, etc. or thecorresponding Greek letter symbols (e.g., α, β, etc.) may sometimes beused.

[0062] The response elements of the present invention can be composed oftwo or more “half sites”, wherein each half site comprises the sequence-RGBNNM-, with the proviso that at least 4 of the nucleotides in thehalf-site sequence are identical with the nucleotides at comparablepositions of the sequence -AGGTCA-. Where one of the half sites variesby 2 nucleotides from the preferred sequence of -AGGTCA-, it ispreferred that the other half site of the response element be the sameas, or vary from the preferred sequence by no more than 1 nucleotide. Itis presently preferred that the 3′-half site (or downstream half site)of a pair of half sites vary from the preferred sequence by at most 1nucleotide.

[0063] Exemplary response elements contemplated by the present inventionare derived from various combinations of half sites having sequencesselected from, for example, -AGGTCA-, -GGTTCA-, -GGGTTA-, -GGGTGA-,-AGGTGA-, -GGGTCA-, and the like.

[0064] The spacer nucleotide sequence employed in the invention responseelements can be any combination of C, T, G, or A.

[0065] Exemplary response elements contemplated by the present inventioninclude:

[0066] 5′-AGGTCA-AGG-AGGTCA-3′,

[0067] 5′-GGGTGA-ATG-AGGACA-3′,

[0068] 5′-GGGTGA-ACG-GGGGCA-3′,

[0069] 5′-GGTTCA-CGA-GGTTCA-3′,

[0070] 5′-AGGTCA-CAGG-AGGTCA-3′,

[0071] 5′-AGGTGA-CAGG-AGGTCA-3′,

[0072] 5′-AGGTGA-CAGG-AGGACA-3′,

[0073] 5′-GGGTTA-GGGG-AGGACA-3′,

[0074] 5′-GGGTCA-TTTC-AGGTCC-3′,

[0075] 5′-AGGTCA-CCAGG-AGGTCA-3′,

[0076] 5′-AGGTGA-ACAGG-AGGTCA-3′,

[0077] 5′-GGTTCA-CCGAA-AGTTCA-3′,

[0078] 5′-GGTTCA-CCGAA-AGTTCA-3′,

[0079] 5′-AGGTCA-CTGAC-AGGGCA-3′,

[0080] 5′-GGGTCA-TTCAG-AGTTCA-3′,

[0081] 5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCAGCTT-3′,5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATAGCTT-3′,5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATATATTAGCTT-3′, and the like.

[0082] Presently preferred response elements contemplated by the presentinvention include:

[0083] 5′-AGGTCA-AGG-AGGTCA-3′,

[0084] 5′-AGGTCA-CAGG-AGGTCA-3′,

[0085] 5′-AGGTGA-CAGG-AGGTCA-3′,

[0086] 5′-AGGTCA-CCAGG-AGGTCA-3′,

[0087] 5′-AGGTGA-ACAGG-AGGTCA-3′, and the like. These are especiallypreferred because they represent synthetic sequences which have not beenobserved in nature, and thus are applicable to a wide variety ofreporter systems (i.e., the use of these response elements will not belimited due to any species preference based on the source of thesequence).

[0088] With respect to the promoter which is part of a transcriptionalcontrol region of the invention, practically any promoter may be used,so long as the transcriptional activity of such a promoter can bemodulated by a response element of the present invention (when suitablypositioned upstream from the promoter). Among such promoters areDelta-MTV promoter of mouse mammary tumor virus, Herpes simplexthymidine kinase (tk) promoter, basal Simian virus SV-40 promoter, theDrosophila alcohol dehydrogenase (ADH) promoter, and the like. Presentlypreferred are promoters which require a response element for activity.

[0089] Virtually any protein or polypeptide of interest can be made withcells transformed with an expression vector of the invention. Suchproteins include hormones, lymphokines, receptors or receptor subunits,immunoglobulin chains and the like. Indicator proteins such as LUC, CAT,and β-Gal can also be made.

[0090] Among the types of cells that can be transformed in accordancewith the invention are mammalian cells, avian cells, insect cells, andthe like, such as, for example, CV-1, COS, F9, P19, CHO, HeLa, NIH 3T3,HuTu80, Rat2 fibroblasts, HT1080.T, chick embryo fibroblasts, quail QT6,Drosophila Schneider S2 cells, and the like.

[0091] The invention method for determining the activity of a testcompound as an agonist or antagonist of ligands for members of thesteroid/thyroid superfamily of receptors can be carried out employingstandard assay techniques, as are well known by those of skill in theart. See, for example, Mangelsdorf et al., in Nature 345: 224-229(1990).

[0092] Test compounds contemplated for screening in accordance with theinvention assay methods include any compound which can potentiallyaffect the ability of receptor to modulate transcription activitythrough a response element of the present invention.

[0093] In accordance with a specific embodiment of the presentinvention, wherein it is possible to distinguish whether or notresponsiveness to a ligand for a first member of the steroid/thyroidsuperfamily of receptors occurs via a pathway unique to a first receptor(relative to other member(s) of the superfamily) or via some otherpathway, responsiveness to said ligand via the pathway for the firstreceptor would result in increased amounts of transactivation as afunction of increased expression of said first receptor, whileresponsiveness to said ligand via the pathway for the second receptorwould result in reduced levels of transactivation as a function ofincreased expression of said second receptor (caused by competition bythe second receptor for ligand needed for the activation of the firstreceptor).

[0094] Receptors, assay methods, and other subject matter pertinent tothe subject matter of the present specification may be found in thefollowing references, which are incorporated herein by reference:Commonly assigned U.S. patent application Ser. No. 108,471, filed Oct.20, 1987 and published as PCT International Publication No. WO 88,03168;commonly assigned U.S. patent application Ser. No. 276,536, filed Nov.30, 1988 and published as European Patent Application Publication No. 0325 849; commonly assigned U.S. patent application Ser. No. 370,407,filed Jun. 22, 1989, said Application listing a Budapest Treaty Depositof a plasmid harboring a cDNA encoding a gamma-retinoic acid receptor,said deposit having been made Jun. 22, 1989 and bearing American TypeCulture Collection Accession No. 40623; Zelent et al., Nature 339, 714(1989); Petkovich et al., Nature 330, 444 (1987); Brand et al., Nature332, 850 (1988).

[0095] Because the DNA segments which comprise the response elements ofthe present invention are relatively short, they may be providedsynthetically, that is by synthesizing the response element-containingoligonucleotide on a DNA synthesizer as is known in the art. It isfrequently very desirable to provide restriction endonuclease sites atthe 3′- and 5′-ends of the oligomer, such that the synthetic responseelement may be conveniently inserted into a DNA expression vector at asite upstream from the promoter, whose transcriptional activity is to beenhanced and which drives transcription of the desired gene. As those ofordinary skill in the art will understand, the response elements of thepresent invention, like other response elements, are orientation and,with wide latitude, position independent. Thus, the response elements ofthe present invention are functional in either orientation and may beplaced in any convenient location from about 30 nucleotides upstream toabout 10,000 nucleotides upstream from the promoter to be affected.

[0096] Preferred cells for use with expression systems employingtranscriptional control regions comprising invention response elementare mammalian cells such as COS cells and CV-1 cells. COS-1 (referred toas COS) cells are mouse kidney cells that express SV40 T antigen (Tag);while CV-1 cells do not express SV40 Tag. CV-1 cells are convenientbecause they lack any endogenous glucocorticoid or mineralocorticoid orother known members of the steroid/thyroid superfamily of hormonereceptors, except that they do produce low levels of βRAR. Thus, viagene transfer with appropriate expression vectors comprising aheterologous gene under the control of a transcriptional control regionof the invention, it is possible to convert these host cells intotransformed cells which produce increased quantities of a desiredprotein in response to induction by a ligand for a member of thesteroid/thyroid superfamily of receptors.

[0097] Expression plasmids containing the SV40 origin of replication canpropagate to high copy number in any host cell which expresses SV40 Tag.Thus, expression plasmids carrying the SV40 origin of replication canreplicate in COS cells, but not in CV-1 cells. Although increasedexpression afforded by high copy number is desirable, it is not criticalto the assay systems described herein. The use of any particular cellline as a host is also not critical, although CV-1 cells are presentlypreferred because they are particularly convenient.

[0098] The invention will now be described in detail by reference to thefollowing non-limiting examples.

EXAMPLES Example 1

[0099] The following demonstrates that the sequences in the promoter ofthe mouse RARβ gene confer retinoic acid (RA) responsiveness, and thatthese sequences represent a target specific for the three RA receptorsubclasses (i.e., alpha-, beta-, and gamma-RAR). The RA response element(RARE) does not mediate significant transcriptional activation byestrogen or glucocorticoid, but it does weakly mediate (by about oneorder of magnitude less) the transcriptional activation by vitamin Dreceptor or thyroid hormone receptors (complexed with cognate ligands).

[0100] A mouse liver genomic DNA library (Clonetech) in lambda vectorEMBL3 was screened with the BamHI-SphI fragment of the human RARβ cDNAclone B1-RARe [see Benbrook et al., in Nature 333: 669-672 (1988)] tolocalize the RARE in the RARβ gene. The probe used contains only firstexon sequences, which are unique to the βRAR gene. A clone harboring a20 kb insert was isolated (containing approximately 10 kb of upstreamsequence, the complete first exon, and 10 kb of the first intron), andthe region surrounding the first exon was subcloned and subjected todideoxy sequence analysis. The sequence of the portion of this clonecontaining the first exon and proximal 5′ DNA is shown in FIG. 1, whichrepresents the sequence of the mouse βRAR promoter region and the firstexon. The TATA and -AGGTCA- motifs are underlined; the first exon splicesite is indicated with an arrow.

[0101] The 10 kb upstream region of the genomic fragment isolated asdescribed above was fused in-frame just downstream of the RARβtranslation initiation codon to a β-galactosidase reporter gene (seeFIG. 2a, which presents the in vivo analysis of RARβ RA response elementsequences, following a series of deletions from the 5′-end of thesequence including the β retinoic acid response element. The sequence atthe junction between the mouse RARβ gene and the β-galactosidasereporter gene is as shown. Numbered amino acids correspond to the nativeRARβ translation product. Restriction sites are abbreviated as follows:N=NotI, X=XhoI, K=KpnI, S=SalI, Nh=NheI, Sc=SacII. The dotted linerepresents plasmid sequences).

[0102] RAR-PL-βGAL was introduced into monkey kidney CV-1 cellscotransfected with RAR expression vector. Enzyme activity was inducedupon retinoic acid addition, indicating that this region of genomic DNAcontains a functional promoter which is responsive to retinoic acid.This was accomplished by introducing a SalI restriction site into thegenomic clone at the indicated position by site-directed mutagenesis;the 10 kb genomic fragment was then excised and cloned into theβ-galactosidase vector pLSV [a derivative of pGH101; Herman et al.,Nucleic Acids Research 14:7130 (1986)], modified to contain a SalI siteand a polylinker sequence by oligo addition, to yield RAR-PL-βGAL.

[0103] A series of deletions from the 5′-end of RAR-PL-βGAL reveal thatsequences mediating RA induction reside within the 2 kb NheI-SacIIfragment (see FIG. 2a and the Table below). Subfragments of this regionwere cloned into the enhancer-dependent luciferase reporter plasmidDMTV-LUC, which contains the mouse mammary tumor virus promoter with thenatural GR response elements deleted [see Hollenberg et al., in Cell 55:899-906 (1988). A 183 bp SmaI fragment (see FIG. 1) is able to conferretinoic acid responsiveness to this heterologous promoter in eitherorientation (see data presented in the Table below). Oligonucleotidesequences derived from this region (see FIG. 2b) were then used tofurther define the RA response element, either in DMTV-LUC or DMTV-CAT(see data presented in the Table below). FIG. 2(b) presents sequences ofoligonucleotides including the β retinoic acid response element used inthese experiments. The terminal lower case bases are foreign to the RARβpromoter, and were included to allow insertion into the unique HindIIIsite of the Delta-MTV vector.

[0104] Thyroid hormone response element (TRE) has been shown to mediatetranscriptional activation by transfected RARs in CV-1 cells, whereasnon-transfected cells show no response [see Umesono et al., in Nature336: 262-265 (1988)]. Surprisingly, Delta-MTV-CAT constructs βRE1, βRE2,and βRE3 (see FIG. 2) showed robust RA-dependent induction in theabsence of cotransfected RAR expression vector. Cotransfection ofRAR-alpha expression vector increased induction by only two-fold, whichdemonstrates that CV-1 cells express a low level of endogenous RAreceptor that is sufficient for efficient activation of vectorscontaining the βRE, but apparently below a threshold for activation ofthe previously studied TREs. A survey of the following cell linesindicated that all were able to efficiently transactivate the βRARE inan RA-dependent fashion in the absence of transfected RAR expressionvector: CV-1, F9 and P19 (mouse teratocarcinomas), CHO, HeLa, NIH 3T3,Rat2 fibroblasts, HT1080.T (human lymphoid), chick embryo fibroblasts,and quail QT6 cells. No cell line has yet been tested which does notexpress this activity.

[0105] Inspection of the sequences of βRE1, βRE2, and βRE3 (see FIG. 2b)identifies a tandem repeat of the 6 bp motif. The center to centerseparation of 11 bp between these repeats is one turn of the DNA helix.Constructs containing single copies of either the 5′- or 3′-half site(βRE4 and βRE5) are functional only upon cotransfection of RARexpression vectors (see FIG. 2d). Not only does this indicate that theRARE is a bonafide target of all three RAR subtypes expressed fromcloned cDNA, but it also demonstrates that these half sites can serve asa minimal RA response element in the context of the Delta-MTV promoter.Apparently, a single half site element of the RARβ gene wouldreciprocally be responsive to the TR, ER, and/or other members of thereceptor superfamily. Cotransfection of the ER, GR, in CV-1 cells withconstruct βRE1 failed to result in appreciable activation in the absenceor following addition of the appropriate ligand, although cotransfectionwith TR and vitamin D receptor (VD3R) CV-1 cells with construct βRE1 didweakly (about 10- to 20-fold less) activate their cognate responseelements.

[0106] Five μg of each of the constructs indicated in the Table weretransfected into CV-1 cells with either RSV-LUC or RSV-βGAL to normalizetransfection efficiencies. Transfections also included RARα expressionvector. Each value in the Table represents duplicate measurements ofplates treated with 10⁻⁷M RA (βGAL experiments) or 10⁻⁶mRA (luciferaseexperiments) relative to plates treated with solvent only. The 183 SmaIrestriction fragment (shown in FIG. 1) was inserted either in theforward (F) or reverse (R) orientation relative to the Delta-MTVpromoter. The (NR) construct contains a 45 bp oligo sequence located 24bp 3′ of βRE1 in the RARβ promoter which was nonresponsive to RA.

[0107] Plasmids were transfected into CV-1 cells and assayed forβ-galactosidase activity either without or with the addition of 10⁻⁷MRA. Negative responses were two-fold induction or less; positiveinductions were seven-fold or greater.

[0108] Cells were transfected in 10 cm dishes with 10 μg of DNAcontaining 5 μg of reporter plasmid, 1-2 μg of either RSV-LUC (a), orRSV-βGAL or pCH110 (c and d), pGEM4 as carrier DNA, and for theexperiments shown in a and d, 1 μg of RSV-RAR expression vector or thesame amount of an RSV vector generating a nonsense transcript. Cellswere harvested 1 day after addition of retinoic acid. All CAT assaysrepresent equivalent amounts of β-galactosidase activity; β-GAL assayswere normalized to luciferase activity. TABLE Retinoic acid inducibilityof reporter constructs Construct Fold increase RAR-PL-βGAL 14RAR-DXN-βGAL 22 RAR-DNhSc-βGAL 2 DMTV-LUC 2 DMTV-Sma183F-LUC 10DMTV-Sma183R-LUC 9 DMTV-LUC 2 DMTV-(NR)-LUC 2 DMTV-βRE1-LUC 14

Example 2

[0109] Receptor expression plasmids used in the following cotransfectionassays have been described previously (pRShTRβ [see Thompson, et al.,Proc. Natl. Acad. Sci. U.S.A. 86: 3494-3498 (1989)]; pRShRARα (seeGiguere, et al., Nature 330: 624-629 (1987)]; pRShRARβ and pRshRARγ [seeIshikawa, et al., Mol. Endocrinol, 4: 837-844 (1990)]; and pRShVDR,[Schule, et al., Cell 61: 497-504 (1990)]. A basal reporter plasmidΔSV-CAT was constructed by replacing the TK promoter in TK-CAT [Damm, etal., Nature 339: 593-597 (1989)] with the SphI-HindIII fragment of theSV40 early promoter. All of the recombinant CAT reporter plasmids usedherein harbor a single copy of the indicated oligonucleotides at theunique HindIII site upstream of the SV40 promoter. Identity of theinserted oligonucleotides was confirmed by sequencing. To improveproduction of receptor proteins in COS cells, a new eukaryoticexpression vector pCMX was prepared by modifying the plasmid CDM8 [Seed,B., Nature 329: 840-842 (1987)]. The CDM8 was cut with MluI and StuI inorder to release the DNA fragment encoding the CMV/T7 promoter, SV40small t intron/poly A signal, polyoma virus enhancer/origin, and SV40enhancer/origin. The resulting fragment was ligated to a larger fragmentof PvuII-digested PUC19. An internal deletion was introduced betweenunique BamHI and BclI sites present in the DCM8 portion. The stuffersequence flanked by XbaI sites was replaced with a synthetic polylinkercoding for 5′-KpnI/Asp718-EcoRV-BamHI-MscI-NheI-3′, followed by astretch of 5′-TAGGTAGCTAG-3′ which can function as a universaltermination signal for protein translation. The coding sequence of theluciferase [de Wet, et al., Mol. Cell. Biol. 7: 725-737 (1987)] andhuman TRβ, RARα, and VDR was placed in the polylinker region of thepCMX, generating pCMX-LUC, pCMX-hTRβ, pCMX-hRARα, and pCMX-hVDR,respectively. The translation start site of the RARα was modified toACCACCATG by attaching the synthetic linker encoding a consensustranslation start signal [Hollenberg, et al., Cell 55: 899-906 (1988)].This modification resulted in much better yield of the receptortranslation as judged in the in vitro-reticulocyte lysate translationsystem.

[0110] For cotransfection assays, a monkey kidney cell line CV-1 waskept in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%charcoal-resin double split calf bovine serum. Transfections wereperformed via calcium-phosphate precipitation method as described byUmesono, et al. in Cell 57: 1139-1146 (1989), with 0.5 μg of a pRSreceptor expression plasmid, 1.0 μg of a reporter CAT plasmid, 5 μg ofpRAS-βGAL [Umesono, et al., Cell 57: 1139-1146 (1989)] as an internalcontrol, and 8.5 μg of carrier plasmid pUCl9. The cells were transfectedfor 8 hours; after washing the DNA precipitates, the cells were thenincubated for an additional 36 hours with or without the ligand (T₃, 100nM; RA, 1 μM; 1,25-(OH)₂ vitamin D₃, 100 nM). Cell extracts wereprepared for βGAL and CAT assays, as described by Umesono, et al.,supra. Transfection of F9 teratocarcinoma cells was carried outemploying a similar method except the cells were incubated for 12 hoursin the presence of the DNA precipitates, and the RA was added at 1 μMfor another 24 hours before harvesting the cells. 2, 5, and 4 μg of thereporter, pRAS-βGAL, and pUCl9 were used, respectively.

[0111] For DNA binding assays, COS cells were cultured in DMEM with 10%calf bovine serum and transfected by the calcium phosphate method with20 μg of the pCMX receptor expression plasmid for 6 hours followed by aglycerol shock. After incubating the transfected COS cells for another48 hours, the cells were harvested to prepare extracts for the DNAbinding assay, carried out as described by Damm et al., Nature 339:593-597 (1989). The extracts were made in 20 mM HEPES (pH 7.4), 0.4 MKCl, 2 mM DTT and 20% glycerol. A similar method was employed to preparea whole cell extract from F9 stem cells. For the binding, 5 μg (COS) or10 μg (F9) of proteins was incubated on ice for 20 minutes, first in 20mM HEPES, 80 mM KCl, 1 mM DTT, 0.1% NP40, 2.5 μg of poly dI/dC, and 10%glycerol (cold competitor oligonucleotides, when included, were addedduring this pre-incubation period). Then 40 fmole of ³²P-labeledoligonucleotide probe (1-2×10⁵ cpm, prepared by filling-in reactionusing Klenow polymerase in the presence of α-³²P-dCTP) was added to thereaction mixture, followed by incubation at room temperature for 30minutes. The receptor-DNA complexes were resolved by electrophoresisthrough 5% polyacrylamide gel containing 5% glycerol at 6 V/cm at roomtemperature. Under the conditions employed, inclusion of the ligand didnot alter the DNA binding pattern of the receptor proteins.

[0112] It has previously been shown that a palindromic TRE (TREp)mediates both T₃ and retinoic acid response, suggesting that the TR andRAR may regulate overlapping sets of genes [Umesono, et al., Nature 336:262-265 (1988)]. To test whether coregulation is a general feature ofTREs, the properties of the T₃-sensitive MHC gene promoter wereexamined. Accordingly, a reporter plasmid covering a 168 bp segment ofthe MHC a gene promoter (αTRE-CAT; Izumo, et al., Nature 334: 539-542(1988)] was cotransfected into CV-1 cells together with expressionplasmids for the human RAR α, β, or γ [Giguere, et al., Nature 330:624-629 (1987); Ishikawa, et al., Mol. Endocrinol 4: 837-844 (1990)] orthe human TRβ [Thompson, et al., Proc. Natl. Acad. Sci. U.S.A. 86:3494-3498 (1989)]. In the absence of the receptor expression plasmids,no stimulation of the CAT enzyme activity was observed by RA or T₃.Expression of TRβ, however, conferred a significant T₃ responseconsistent with previous result employing the rat TRα (Izumo, et al.,supra). On the other hand, production of any one of three isoforms ofthe RARs failed to promote an RA response although similar CAT reporterconstructs encoding TREp are responsive to both TRs and RARs.

[0113] To understand the molecular basis of this phenotypic differencebetween the TREp and the MHC-TRE, a set of CAT reporters based on aminimal SV40 early promoter (ΔSV-CAT) was prepared. Co-transfectionexperiments with the TRβ expression vector demonstrated that promotersharboring either the TREp or the wild-type MHC sequence (MHC-L) areresponsive to T₃ (see FIG. 3a).

[0114] Target hormone response elements described in FIG. 3 weresynthesized as double-stranded oligonucleotides with an overhangingtetranucleotide (5′-agct-3′) at both ends. A single copy of theseoligonucleotides was cloned at the unique HindIII site present in thebasal promoter CAT construct ΔSV-CAT. Capitalized portions in thenucleotide sequences correspond to those found in the natural promotersexcept TREp and rGH21, which are synthetic. Bold letters indicate the“AGGTCA” motif and “X” denotes a nucleotide substitution from thismotif. Numbers between the arrows are the size of the spacer and thosein columns represent the fold inductions of the CAT enzyme activitystimulated by the hormones in either the TRβ (±T₃ at 100 nM) or RARα(±RA at 1 μM) producing CV-1 cells. Inductions observed on the basalconstruct ΔSV-CAT by T₃ and RA are 0.8 and 1.4 fold, respectively.

[0115] In FIG. 3a, the TREp referred to is an optimized palindromic ratgrowth hormone TRE (see Glass et al., supra) which stands also as anefficient RARE (see Umesono et al., supra). MHC-L encodes a TRElocalized at positions -XXX and -YYY in the MHC gene promoter (see Glasset al., supra). MHC-S and MHC-D contain deletion(s), indicated bybrackets, from the MHC-L.

[0116] Because the MHC-L resembles the rat growth hormone TRE and TREp,it has been proposed that the actual response element is composed of apartial palindrome of AGGTCA [see FIG. 3a; see also Izumo, et al.,supra, and Glass, et al., Cell 59: 697-708 (1989)]. However, arestricted region corresponding to the predicted palindrome (MHC-Sreporter) failed to confer T₃ responsiveness, indicating that thisproposal is incorrect. In contrast, a segment of the MHC sequence(MHC-D) including an adjacent sequence, resulted in a recovery of thefull response, delineating a minimal boundary for the MHC-TRE. Anexamination of this sequence surprisingly fails to reveal a palindrome,but instead identifies a direct (i.e., tandem) repeat of the hexamersAGGTGA and AGGACA.

[0117] While each individual half-site resembles the AGGTCA motif inTREp, the orientation of the half sites in the MHC-TRE is distinct. Theintriguing ability of this element to respond to T₃ and its failure torespond to RA suggested that detailed exploration of the properties ofthe sequence might reveal additional detail about hormone response. Asshown in FIG. 3b, MHC-N encodes a 23-nucleotide core of the wild-typesequence (MHC-TRE). M1 and M3 contain specific nucleotide substitutionsin the proposed direct repeat motif (denoted by shadowed letters and “X”in the arrow), while M2 mutant instead carries specific nucleotidesubstitutions in the presumed palindrome. Using COS cell extractsprepared in the presence or absence of the TRβ expression plasmid, itwas possible to detect specific binding of the TRβ protein to the³²P-labeled MHC-N oligonucleotides by a gel retardation assay. As acompetitor, a 50-fold excess of unlabeled oligonucleotides were added tothe binding reactions; both MHC-N and M2 competed, but M1 and M3 didnot. This clearly demonstrates that the two G residues in both “AGGTGA”and “AGGACA” are necessary to constitute the TRβ binding site. Thecentral three nucleotides AGG (that are part of the putative palindrome)are changeable without a significant impact on the receptor binding.This is consistent also with binding data obtained by an avidin-biotinDNA binding assay and a foot-printing pattern on the MHC α gene promoterwith the TRs [Flink, et al., J. Biol. Chem. 265: 11233-11237 (1990)]. Asexpected, cotransfection assays with each construct reveal that both M1and M3 are completely silent to T₃ whereas the M2 reporter retains a T₃response though impaired (see FIG. 3b). These observations support theview that the MHC-TRE is not palindromic, but instead consists of adirect repeat of “AGGTCA”-like half-sites separated by 4 nucleotides.

[0118] In view of the results with the MHC-TRE, other previouslycharacterized TREs were similarly re-examined. In a recent report byPetty, et al., in J. Biol. Chem. 265: 7395-7400 (1990), a short 20 bpDNA segment found in the T₃ responsive malic enzyme gene promoter wasfootprinted by the TR. When this sequence is placed in the minimal SV40promoter it confers T₃ responsiveness, and based on the above model,contains a direct repeat motif (see FIG. 3c). In the Figure, malicenzyme TRE corresponds to -XXX to -YYY from a transcription start site(see Petty et al., supra). A thyroid response element found in themurine Maloney Leukemia Virus (MLV) LTR (by both receptor binding and invivo function; “MLV-LTR”740 is taken from Sap et al. [see rat growthhormone gene TRE [see Brent et al., J. Biol. Chem. 264: 178-182 (1989)].βRARE corresponds to βRE2 reported in Sucov et al. [Proc. Natl. Acad.Sci. U.S.A. 87: 5392-5398 (1990)].

[0119] When the TRE found in the MLV-LTR is placed into the SV40reporter it confers T₃ inducibility. Based on the MHC model, a directrepeat of the “AGGTCA”-like sequence can be identified in thesesequences (see FIG. 3c). In contrast, a rat growth hormone (rGH) genesequence proposed by Brent, et al. supra, as a candidate TRE, fails toconfer inducibility to the SV40 reporter (see FIG. 3c). However, asshown below, this sequence fails to interact with the TR in vitro andthus, may not be a bona fide response element.

[0120] While both the TRs and RARs are able to bind palindromic TREs[see Umesono, et al., Nature 336: 262-265 (1988); Glass, et al., Cell54: 313-323 (1988); and Graupner, et al., Nature 340: 653-656 (1989)],the direct repeat TRE is TR specific. To understand the molecular natureof this restriction, the structure of previously characterized naturalRAREs was re-evaluated. Recently, one such sequence was identified inthe promoter region of the mouse [Sucov, et al., and human RARβ genes[de The, et al., Nature 343: 177-180 (1990)]. Interestingly, this RARE(designated as βRARE) is composed of a direct repeat motif and isselectively activated by the RAR but not by the TR (Sucov, et al.,activated by the RAR but not by the TR (Sucov, et al., supra). To betterunderstand the basis of exclusive recognition and selective response ofthe direct repeat TRE and RARE motifs, a series of comparative in vitroDNA-binding and functional assays were conducted.

[0121] Oligonucleotides listed in FIG. 3 were used as a probe to testspecific DNA-binding of the RARα and TRβ in a gel retardation assay. Onthe βRARE, a faint background binding activity was detected in the mocktransfected COS cell extract. The TRβ extract contained a weak, yetdetectable binding activity over the background; however, the signalobtained with the RARα extract was dramatically enhanced. In addition tothe MHC-TRE, the malic enzyme TRE and MLV-LTR TRE are all high affinityTRβ binding sites, consistent with the previous observations of Glass,et al. [Cell 59: 697-708 (1989)], Sap, et al., [Nature 340: 242-244(1989], Petty, et al. [J. Biol. Chem. 265: 7395-7400 (1990)], and Flink,et al. [J. Biol. Chem. 265: 11233-11237 (1990)]. On the other hand, TRβbinds very poorly to the rGH21 probe, which (as described above) alsofails to confer T₃-dependent transactivation in transfection experiments(FIG. 3c). Finally, no appreciable binding of the RARα protein was seenwith any of these TREs. In each case the result obtained from in vivotransactivation and in vitro DNA binding are consistent. Thus, thesetandem HREs possess intrinsic differences to impart selective TR or RARspecific binding and activation, and virtually eliminate the possibilityof hormonal cross-talk.

[0122] Starting from the MHC-TRE (MHC-N), a set of oligonucleotides (seeFIG. 4a) were designed to introduce variations in the half-site spacing(MCH+1), the half-site sequence (MHC-T), or both (MHC-R). A single copyof these oligonucleotides was placed in the basal reporter constructΔSV-CAT (designated by “(−)” in FIG. 4b), giving rise to a set of CATreporter plasmids together with one encoding TREp. Parallelcotransfections with expression plasmids for TRβ or RARα were performedin CV-1 cells along with the ΔSV-CAT reporter containing a single copyof the variant HRE. After addition of hormone (RA, 1 μM; T₃, 100 nM),the cell extracts were assayed for the CAT activity by measuring theβ-galactosidase activity produced by cotransfected pRAS-βGAL. In thefigure, numbers in the columns indicate the level of induction of CATactivity by the hormone.

[0123] Using the control palindromic TREp reporter, TRβ and RARαelicited a 14 and 9 fold induction, respectively, while the MHC-Nconfers T₃ but not RA response. By increasing the spacing by onenucleotide, the T₃ response decreases from 10 fold to less than 2 fold,and conversely confers a modest but significant induction by RA (4fold). In MHC-T the “AGGACA” half-site was corrected to “AGGTCA”,resulting in a better T₃ response (15 fold) than the wild type (10fold), while producing only a marginal RA response (2.6 fold). Incontrast, increasing the half-site spacing of MHC-T by one nucleotide(to MHC-R) generates an efficient RARE (7.4 fold RA induction), whilereciprocally blunting T₃ responsiveness (1.3 fold). The position of thenucleotide insertion within the spacer is flexible; the phenotype ofanother mutant carrying the MHC-T mutation together with an additional Gin the middle of the spacer (CAGG to CAGGG) is identical to that of theMHC-R. Thus, half-site spacing is indicated as the determinativeparameter since a single nucleotide insertion (MHC-T to MHC-R) issufficient to interconvert a TRE to an RARE.

[0124] Using the mobility shift assay, the DNA-binding capacity ofextracts from COS cells expressing TRβ and RARα to these sequences wasalso tested. The positive TREs (TREp, MHC-N, and MHC-T) showed efficientcompetition with MHC-T notably better than the wild type. In contrast,the RA response elements (MHC+1, MHC-R, and βRARE) are poor competitors.This pattern is consistent with that of the in vivo activation (FIG. 4).A reciprocal pattern was obtained when the same oligonucleotides wereused to compete RARα binding to the ³²P-labeled βRARE. Thus, RA responseelements (TREp, βRARE, and MHC-R) are all effective competitors, whileMHC-TRE fails to compete at all. Furthermore, MHC-T, which differs byonly one nucleotide from MHC-R, also fails to compete efficiently. Thisprovides a striking example of the role of half-site spacing ingenerating a functional response element. The comparable bindingaffinity of the MHC-R to that of TREp, together with much weakeraffinity of MHC+1 and MHC-T, correlate well with the results from thetransfection assays (FIG. 4).

[0125] The parallels between in vivo transactivation and in vitroDNA-binding patterns by two distinct receptors strongly supports theconclusion that the TR binds to an “AGGTCA”-like direct repeat with a4-nucleotide spacer while the RAR recognizes the similar motif but witha spacing of 5 nucleotides.

[0126] Recent characterization of a VDRE found in the rat osteocalcingene promoter [rOST-VDRE; see Demay, et al., J. Biol. Chem. 266:1008-1013 (1990); and Markose, et al., Proc. Natl. Acad. Sci. U.S.A. 87:1701-1705 (1990)] revealed the presence of three “AGGTCA” relatedsequences in a close proximity (5′-CTGGGTGAATGAGGACATTACTGACC-3′). The5′ portion of the rOST-VDRE contains a tandem repeat of “GGGTGA” and“AGGACA” and is similar to the MHC-TRE (FIG. 3). Because of theimportant role of the half-site spacing for TR and RAR selectivity, itwas tested to see if this observation might extend to the VDRE in whichhalf-sites are spaced by 3 nucleotides.

[0127] Accordingly, a nested set of synthetic hormone response elementswere designed by making a direct repeat of “AGGTCA” with a spacer sizevariation of 3, 4, or 5 residues (FIG. 5a). DR-3, DR-4, and DR-5 eachcode for perfect tandem repeats of the “AGGTCA” hexamers (indicated byarrows in the figure), separated by 3, 4, and 5 nucleotides,respectively. GDR-3, GDR-4, and GDR-5 are identical to the DRoligonucleotides except that the half-site sequence was changed to“AGAACA”, a GRE half-site.

[0128] Since DR-4 encodes two copies of “AGGTCA” in direct repeatseparated by four nucleotides, it should, in principle, be a TRE. Asingle nucleotide insertion to create DR-5, with a 5-nucleotide spacer,should be an RARE. Similarly, one nucleotide deletion from the spacergives rise to DR-3, a VDRE candidate. Oligonucleotides were alsosynthesized which share identical structures to the DR series, butencode a GRE half-site (AGAACA) instead of AGGTCA, as a further controlfor sequence specificity (i.e., the GDR series; see FIG. 5a).

[0129] The above strategies were used to test both in vivotransactivation and in vitro DNA-binding of these artificial HREs forTRβ, RARα, and VDR. A single copy of the DR or GDR oligonucleotides wascloned at the unique HindIII site present in the basal promoter-CATconstruct, ΔSV-CAT, giving rise to DR-3-CAT, DR-4-CAT, DR-5-CAT,GDR-3-CAT, GDR-4-CAT, and GDR-5-CAT reporters. One μg of the indicatedreporters [“(−)” refers to ΔSV-CAT] were cotransfected into CV-1 cellswith 0.5 μg of an expression plasmid for VDR, TRβ, or RARα. After 36hours of incubation with the cognate ligands (VD₃ and T₃, 100 nM; RA, 1μM), the cells were harvested for CAT assay after normalization withβ-galactosidase activity produced from the cotransfected controlreporter pRAS-βGAL. The CAT activity obtained through ΔSV-CAT in theabsence of ligand was taken as 1 for each of the receptors.

[0130] Transfection assays in CV-l cells revealed that DR-3 is indeed anovel vitamin D3 response element (8.3 fold induction, see FIG. 5b). Aspredicted, based on the above-described model, a single nucleotideinsertion in the spacer from DR-3 (to produce DR4) mutuallyinterconverts the vitamin D3 and T₃ responses (22 fold induction by T₃through DR-4, FIG. 5c). The half-site mutants GDR-3 and GDR-4 arecompletely inactive for both the VDR and TR. While DR-5 confers the bestRA response (9 fold induction), DR-3 and DR-4 show clear activity (FIG.5d). As shown below, this is probably a consequence of overexpression ofthe receptor protein. These properties of the synthetic DR hormoneresponse elements can be transferred to a different basal promoter suchas Herpes simplex virus thymidine Kinase gene promoter.

[0131] When the DR reporters are transfected into RA responsive F9teratocarcinoma stem cells, only DR-5 serves as a functional RARE.Similarly, when the same set of reporters as in FIG. 4 were transfectedinto F9 cells, only MHC-R (5-nucleotide spacer) and TREp are potentRAREs. In contrast, its RARE antecedents (MHC-N, MHC+1, MHC-T) areinert. Using βRARE as a probe, a specific protein-DNA complex wasdetected in extracts prepared from the F9 stem cells, which reveals aDNA-binding pattern identical to that of RARα-transfected COS cellextracts.

[0132] Finally, in vitro DNA-binding assays were carried out with COScell extracts containing one of these receptor proteins. With the DR-3,DR-4, and DR-5 as a labeled probe, a specific protein-DNA complex wasobserved, which is dependent on the expression of the VDR, TRβ, and RARαprotein, respectively, in the cell. These studies parallel thetransactivation experiments and indicate that the DR-3, DR-4 and DR-5sequences represent specific binding response elements for the VDR, TRβ,and RARα, respectively.

[0133] While the invention has been described in detail with referenceto preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed herein.

1 51 1 900 DNA Mus sp. CDS (633)..(788) Mouse beta-RAR promoter region 1ccgcggcgct ggctgaaggc tcttgcaggg gtgctgggag tttttaagcg ctgtgagaat 60cctgggagtt ggtgatgtca gactggttgg gtcatttgaa ggttagcagc ccgggaaggg 120ttcaccgaaa gttcactcgc atatattagg caattcaatc tttcattccg tgtgacagaa 180gtggtaggaa gtgagctgct ccgaggcagg agggtctatt ctttgtcaaa gggggggacc 240agagttcccg tgcgccgcgg ccacaagact gggatgcaga ggacgcgagc cacccgggca 300gggagcgtct gggcaccggc ggggtaggac ccgcgcgctc ccggagcctg cgcgggcgtc 360gcctggaagg gagaacttgg gatcggtgcg ggaacccccg ccctggctgg atcggccgag 420cgagcctgga aaatggtaaa tgatcatttg gatcaattac aggcttttag ctggcttgtc 480tgtcataatt catgattcgg ggctgggaaa aagaccaaca gcctacgtgc caaaaaaggg 540gcagagtttg atggagttcg tggacttttc tgtgcggctc gcctccacac ctagaggata 600agcatctttg cagagcgcgg tgcggagaga tc atg ttt gac tgt atg gat gtt 653 MetPhe Asp Cys Met Asp Val 1 5 ctg tca gtg agt ccc ggg cag atc ctg gat ttctac acc gcg agc cct 701 Leu Ser Val Ser Pro Gly Gln Ile Leu Asp Phe TyrThr Ala Ser Pro 10 15 20 tcc tcc tgc atg ctg cag gaa aag gct ctc aaa gcctgc ctc agt gga 749 Ser Ser Cys Met Leu Gln Glu Lys Ala Leu Lys Ala CysLeu Ser Gly 25 30 35 ttc acc cag gcc gaa tgg cag cac cgg cat act gct caatgtaggttta 798 Phe Thr Gln Ala Glu Trp Gln His Arg His Thr Ala Gln 40 4550 tttttttttt tcctttcttt taccaaggaa aaataaatgt ctctcttgca tgcaataaag 858acactggaat aaagtgcagt ggtggcaaga caaagggttt aa 900 2 52 PRT Mus sp.Mouse beta-RAR promoter region 2 Met Phe Asp Cys Met Asp Val Leu Ser ValSer Pro Gly Gln Ile Leu 1 5 10 15 Asp Phe Tyr Thr Ala Ser Pro Ser SerCys Met Leu Gln Glu Lys Ala 20 25 30 Leu Lys Ala Cys Leu Ser Gly Phe ThrGln Ala Glu Trp Gln His Arg 35 40 45 His Thr Ala Gln 50 3 27 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide,Beta-Retinoic Acid Response Element 3 tgt atg gat gtg tcg acc gga tccatg 27 Cys Met Asp Val Ser Thr Gly Ser Met 1 5 4 9 PRT ArtificialSequence Description of Artificial Sequence Amino acid, Beta-RetinoicAcid Response Element 4 Cys Met Asp Val Ser Thr Gly Ser Met 1 5 5 43 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide,Beta-Retinoic Acid Response Element 1 5 aagcttaagg gttcaccgaa agttcactcgcatatattag ctt 43 6 38 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide, Beta-Retinoic Acid Response Element 2 6aagcttaagg gttcaccgaa agttcactcg catagctt 38 7 34 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide,Beta-Retinoic Acid Response Element 3 7 aagcttaagg gttcaccgaa agttcactcagctt 34 8 28 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide, Beta-Retinoic Acid Response Element 4 8 aagcttcgaaagttcactcg catagctt 28 9 23 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide, Beta-Retinoic Acid Response Element5 9 aagcttaagg gttcaccgag ctt 23 10 25 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide, TRE-p 10 agcttcaggtcatgacctga gagct 25 11 38 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide, MHC-L 11 agctccttgg ctctggaggtgacaggagga cagcagct 38 12 25 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide, MHC-S 12 agctctctgg aggtgacaggaagct 25 13 28 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide, MHC-D 13 agcttgaggt gacaggagga cagcagct 28 1429 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide, MHC-N 14 agctggaggt gacaggagga cagcaagct 29 15 29 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide,M1 15 agctggaaat gacaggagga cagcaagct 29 16 29 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide, M2 16 agctggaggtgacgaaagga cagcaagct 29 17 29 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide, M3 17 agctggaggt gacaggaaaacagcaagct 29 18 28 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide, malic enzyme 18 agctggggtt aggggaggac agtaagct28 19 30 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide, MLV-LTR 19 agctcagggt catttcaggt ccttgaagct 30 20 26DNA Artificial Sequence Description of Artificial SequenceOligonucleotide, rGH21 20 agctaggtaa gatcaggtaa gtagct 26 21 36 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide,Beta-RARE 21 agcttaaggg ttcaccgaaa gttcactcgc atagct 36 22 30 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide,MHC+1 22 agctggaggt gaacaggagg acagcaagct 30 23 29 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide, MHC-T 23agctggaggt gacaggaggt cagcaagct 29 24 30 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide, MHC-R 24 agctggaggtgaacaggagg tcagcaagct 30 25 28 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide, DR-3 25 agcttcaggt caaggaggtcagagagct 28 26 28 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide, GDR-3 26 agcttcagaa caaggagaac agagagct 28 2729 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide, DR-4 27 agcttcaggt cacaggaggt cagagagct 29 28 29 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide,GDR-4 28 agcttcagaa cacaggagaa cagagagct 29 29 30 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide, DR-5 29agcttcaggt caccaggagg tcagagagct 30 30 30 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide, GDR-5 30 agcttcagaacaccaggaga acagagagct 30 31 27 DNA Artificial Sequence Description ofArtificial Sequence Substantially pure DNA response element 31rgbnnmnnnn nnnnnnnnnn nrgbnnm 27 32 27 DNA Artificial SequenceDescription of Artificial Sequence Substantially pure DNA responseelement 32 aggtcannnn nnnnnnnnnn naggtca 27 33 71 PRT ArtificialSequence Description of Artificial Sequence Highly Conserved Amino Acidsof the DNA-Binding Domain of Members of the Superfamily 33 Cys Xaa XaaCys Xaa Xaa Asp Xaa Ala Xaa Gly Xaa Tyr Xaa Xaa Xaa 1 5 10 15 Xaa CysXaa Xaa Cys Lys Xaa Phe Phe Xaa Arg Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 Xaa XaaXaa Lys Xaa Xaa Arg Xaa Xaa Cys Xaa Xaa Cys Arg Xaa Xaa 50 55 60 Lys CysXaa Xaa Xaa Gly Met 65 70 34 15 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide; DNA response element 34 aggtcaaggaggtca 15 35 15 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide; DNA response element 35 gggtgaatga ggaca 15 3615 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide; DNA response element 36 gggtgaacgg gggca 15 37 15 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide;DNA response element 37 ggttcacgag gttca 15 38 16 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide; DNAresponse element 38 aggtcacagg aggtca 16 39 16 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide; DNA response element39 aggtgacagg aggtca 16 40 16 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide; DNA response element 40 aggtgacaggaggaca 16 41 16 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide; DNA response element 41 gggttagggg aggaca 1642 16 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide; DNA response element 42 gggtcatttc aggtcc 16 43 17 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide;DNA response element 43 aggtcaccag gaggtca 17 44 17 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide; DNAresponse element 44 aggtgaacag gaggtca 17 45 17 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide; DNA response element45 ggttcaccga aagttca 17 46 17 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide; DNA response element 46 aggtcactgacagggca 17 47 17 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide; DNA response element 47 gggtcattca gagttca 1748 11 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide; DNA response element 48 taggtagcta g 11 49 26 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide;DNA response element 49 ctgggtgaat gaggacatta ctgacc 26 50 16 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide;DNA response element 50 aggtcccaga aggtca 16 51 17 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide; DNAresponse element 51 aggtcactag gaggtca 17

That which is claimed is:
 1. A method of determining whether a testcompound has agonist activity for a member of the steroid/thyroidsuperfamily of receptors, said method comprising: (a) culturing cellstransformed with a vector for the expression of an indicator protein, inthe presence of a member of the steroid/thyroid superfamily ofreceptors, and in the presence and absence of said test compound,wherein said vector comprises a substantially pure DNA response elementoperatively linked to a promoter which is not normally subject totranscriptional activation and/or repression by ligand for said memberof the steroid/thyroid superfamily and linked operatively fortranscription of a gene encoding said indicator protein, so as to confertranscriptional activation and/or repression activity on said promoterin the presence of a compound having agonist activity, wherein said DNAresponse element comprises at least two half sites, each separated bythree, four, or five spacer nucleotides, wherein each half site isindependently selected from -AGGTCA-, -GGGTTA-, -GGGTGA-, -AGGTGA-,-AGGACA-, -GGGGCA-, or -GGGTCA-, -AGGTCC-, -GGTTCA-, -AGTTCA-, or-AGGGCA-; with the proviso that if three spacer nucleotides separate theat least two half sites, one, but not both, half sites is -GGTTCA-; andif four spacer nucleotides separate the at least two half sites, whenone half site is -AGGTCA-, the other half site is not -AGGTCC-; and iffive spacer nucleotides separate the at least two half sites, one, butnot both, half sites is -AGGTCA-, and when one half site is -AGTTCA-,the other half site is not -GGTTCA-; wherein said three spacernucleotides are selected from -AGG-, -ATG-, -ACG-, or -CGA-; andthereafter (b) comparing the amount of said indicator protein expressedin the presence of said test compound to the amount of said indicatorprotein expressed in the absence of said test compound.
 2. A methodaccording to claim 1, wherein said DNA response element comprises thesequence: 5′-AGGTCA-AGG-AGGTCA-3′ (SEQ ID NO:34).
 3. A method accordingto claim 1, wherein said four spacer nucleotides are selected from-CAGG-, -GGGG-, or -TTTC-.
 4. A method according to claim 3, whereinsaid DNA response element comprises the sequence:5′-AGGTCA-CAGG-AGGTCA-3′ (SEQ ID NO:38), or 5′-AGGTGA-CAGG-AGGTCA-3′(SEQ ID NO:39).
 5. A method according to claim 1, wherein said fivespacer nucleotides are selected from -CCAGG-, -ACAGG-, -CCGAA-, -CTGAC-,or -TTGAC-.
 6. A method according to claim 5, wherein said DNA responseelement comprises the sequence: 5′-AGGTGA-ACAGG-AGGTCA-3′ (SEQ IDNO:44), 5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCAGCTT-3′ (SEQ ID NO:7),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATAGCTT-3′ (SEQ ID NO:6), or5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATATATTAGCTT-3′ (SEQ ID NO:5). 7.A method of determining whether a test compound has antagonist activityfor a member of the steroid/thyroid superfamily of receptors, saidmethod comprising: (a) culturing cells transformed with a vector for theexpression of an indicator protein, in the presence of (i) a member ofthe steroid/thyroid superfamily of receptors, and (ii) an agonist forsaid member; and in the further presence and absence of said testcompound, wherein said vector comprises a substantially pure DNAresponse element operatively linked to a promoter which is not normallysubject to transcriptional activation and/or repression by ligand forsaid member of the steroid/thyroid superfamily and linked operativelyfor transcription of a gene encoding said indicator protein, so as toconfer transcriptional activation and/or repression activity on saidpromoter in the presence of said agonist, wherein said DNA responseelement comprises at least two half sites, each separated by three,four, or five spacer nucleotides, wherein each half site isindependently selected from -AGGTCA-, -GGGTTA-, -GGGTGA-, -AGGTGA-,-AGGACA-, -GGGGCA-, or -GGGTCA-, -AGGTCC-, -GGTTCA-, -AGTTCA-, or-AGGGCA-; with the proviso that if three spacer nucleotides separate theat least two half sites, one, but not both, half sites is -GGTTCA-; andif four spacer nucleotides separate the at least two half sites, whenone half site is -AGGTCA-, the other half site is not -AGGTCC-; and iffive spacer nucleotides separate the at least two half sites, one, butnot both, half sites is -AGGTCA-, and when one half site is -AGTTCA-,the other half site is not -GGTTCA-; wherein said three spacernucleotides are selected from -AGG-, -ATG-, -ACG-, or -CGA-; andthereafter (b) comparing the amount of said indicator protein expressedin the presence of said test compound to the amount of said indicatorprotein expressed in absence of said test compound.
 8. A methodaccording to claim 7, wherein said DNA response element comprises thesequence: 5′-AGGTCA-AGG-AGGTCA-3′ (SEQ ID NO:34).
 9. A method accordingto claim 7, wherein said four spacer nucleotides are selected from-CAGG-, -GGGG-, or -TTTC-.
 10. A method according to claim 9, whereinsaid DNA response element comprises the sequence:5′-AGGTCA-CAGG-AGGTCA-3′ (SEQ ID NO:38), or 5′-AGGTGA-CAGG-AGGTCA-3′(SEQ ID NO:39).
 11. A method according to claim 7, wherein said fivespacer nucleotides are selected from -CCAGG-, -ACAGG-, -CCGAA-, -CTGAC-,or -TTGAC-.
 12. A method according to claim 11, wherein said DNAresponse element comprises the sequence: 5′-AGGTGA-ACAGG-AGGTCA-3′ (SEQID NO:44), 5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCAGCTT-3′ (SEQ ID NO:7),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATAGCTT-3′ (SEQ ID NO:6), or5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATATATTAGCTT-3′ (SEQ ID NO:5). 13.A method to distinguish whether or not responsiveness to a ligand for afirst receptor member of the steroid/thyroid superfamily of receptorsoccurs via a pathway unique to said first member of the steroid/thyroidsuperfamily, relative to a second member of said superfamily, saidmethod comprising: (a) contacting an indicator protein expression vectorwith ligand for said first receptor member, and varying ratios of anexpression vectors for said first and second receptor members, whereinsaid indicator protein expression vector comprises a substantially pureDNA response element operatively linked to a promoter which is notnormally subject to transcriptional activation and/or repression byligand for a receptor of the steroid/thyroid superfamily and linkedoperatively for transcription of a gene encoding said indicator protein,so as to confer transcriptional activation and/or repression activity onsaid promoter in the presence of said ligand, wherein said DNA responseelement comprises at least two half sites, each separated by three,four, or five spacer nucleotides, wherein each half site isindependently selected from -AGGTCA-, -GGGTTA-, -GGGTGA-, -AGGTGA-,-AGGACA-, -GGGGCA-, or -GGGTCA-, -AGGTCC-, -GGTTCA-, -AGTTCA-, or-AGGGCA-; with the proviso that if three spacer nucleotides separate theat least two half sites, one, but not both, half sites is -GGTTCA-; andif four spacer nucleotides separate the at least two half sites, whenone half site is -AGGTCA-, the other half site is not -AGGTCC-; and iffive spacer nucleotides separate the at least two half sites, one, butnot both, half sites is -AGGTCA-, and when one half site is -AGTTCA-,the other half site is not -GGTTCA-; wherein said three spacernucleotides are selected from -AGG-, -ATG-, -ACG-, or -CGA-; andthereafter (b) determining the effect of increasing ratios of said firstreceptor member to said second receptor member on modulation oftranscription activity of said response element by said ligand.
 14. Amethod according to claim 13, wherein said DNA response elementcomprises the sequence: 5′-AGGTCA-AGG-AGGTCA-3′ (SEQ ID NO:34).
 15. Amethod according to claim 13, wherein said four spacer nucleotides areselected from -CAGG-, -GGGG-, or -TTTC-.
 16. A method according to claim15, wherein said DNA response element comprises the sequence:5′-AGGTCA-CAGG-AGGTCA-3′ (SEQ ID NO:38), or 5′-AGGTGA-CAGG-AGGTCA-3′(SEQ ID NO:39).
 17. A method according to claim 13, wherein said fivespacer nucleotides are selected from -CCAGG-, -ACAGG-, -CCGAA-, -CTGAC-,or -TTGAC-.
 18. A method according to claim 17, wherein said DNAresponse element comprises the sequence: 5′-AGGTGA-ACAGG-AGGTCA-3′ (SEQID NO:44), 5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCAGCTT-3′ (SEQ ID NO:7),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATAGCTT-3′ (SEQ ID NO:6), or5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATATATTAGCTT-3′ (SEQ ID NO:5). 19.A method to screen a plurality of test compounds to identify thosecompounds which act as ligands for members of the steroid/thyroidsuperfamily of receptors, said method comprising: (a) culturing cellstransformed with (i) a vector for the expression of an indicator proteinand (ii) an expression vector for a member of the steroid/thyroidsuperfamily of receptors, in the presence and absence of said testcompounds, wherein said receptor, in the presence of its cognate ligand,is capable of binding to a substantially pure DNA response element,wherein said DNA response element comprises at least two half sites,each separated by three, four, or five spacer nucleotides, wherein eachhalf site is independently selected from -AGGTCA-, -GGGTTA-, -GGGTGA-,-AGGTGA-, -AGGACA-, -GGGGCA-, or -GGGTCA-, -AGGTCC-, -GGTTCA-, -AGTTCA-,or -AGGGCA-; with the proviso that if three spacer nucleotides separatethe at least two half sites, one, but not both, half sites is -GGTTCA-;and if four spacer nucleotides separate the at least two half sites,when one half site is -AGGTCA-, the other half site is not -AGGTCC-; andif five spacer nucleotides separate the at least two half sites, one,but not both, half sites is -AGGTCA-, and when one half site is-AGTTCA-, the other half site is not -GGTTCA-; wherein said three spacernucleotides are selected from -AGG-, -ATG-, -ACG-, or -CGA-; andthereafter (b) assaying for the modulation of expression of saidindicator protein.
 20. A method according to claim 19, wherein said DNAresponse element comprises the sequence: 5′-AGGTCA-AGG-AGGTCA-3′ (SEQ IDNO:34).
 21. A method according to claim 19, wherein said four spacernucleotides are selected from -CAGG-, -GGGG-, or -TTTC-.
 22. A methodaccording to claim 21, wherein said DNA response element comprises thesequence: 5′-AGGTCA-CAGG-AGGTCA-3′ (SEQ ID NO:38), or5′-AGGTGA-CAGG-AGGTCA-3′ (SEQ ID NO:39).
 23. A method according to claim19, wherein said five spacer nucleotides are selected from -CCAGG-,-ACAGG-, -CCGAA-, -CTGAC-, or -TTGAC-.
 24. A method according to claim23, wherein said DNA response element comprises the sequence:5′-AGGTGA-ACAGG-AGGTCA-3′ (SEQ ID NO:44),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCAGCTT-3′ (SEQ ID NO:7),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATAGCTT-3′ (SEQ ID NO:6), or5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATATATTAGCTT-3′ (SEQ ID NO:5).